Parallelism in neurodegenerative biomarker tests: hidden errors and the risk of misconduct

Biomarkers are critical tools in the diagnosis and monitoring of neurodegenerative diseases. Reliable quantification depends on assay validity, especially the demonstration of parallelism between diluted biological samples and the assay's standard curve. Inadequate parallelism can lead to biased concentration estimates, jeopardizing both clinical and research applications. Here, we systematically review the evidence of analytical parallelism in body fluid (serum, plasma, cerebrospinal fluid) biomarker assays for neurodegeneration and evaluate the extent, reproducibility, and reporting quality of partial parallelism. This systematic review was registered on PROSPERO (CRD42024568766) and conducted in accordance with PRISMA guidelines. We included studies published between December 2010 to July 2024 without language restrictions. Eligible studies included original research assessing biomarker concentrations in body fluids with data suitable for evaluating serial dilution and standard curve parallelism. The data extraction for interrogating parallelism included dilution steps, measured concentrations, and sample types. For each study, we generated parallelism plots in a uniform and comparable way. These graphs were used to come to a balanced decision on whether parallelism or partial parallelism was present. The risk of bias was assessed based on sample preparation, buffer consistency, and methodological transparency. Of 44 eligible studies, 19 provided sufficient data for generating 49 partial parallelism plots. Only 7 plots (14%) demonstrated clear partial parallelism. Partial parallelism was typically achieved over a narrow dilution range of about three doubling steps. Most assays deviated from parallelism, risking over- or underestimation of biomarker levels if determined at different dilution steps. A high risk of bias was identified in 9 studies using spiked or artificial samples, inconsistent dilution buffers, or incomplete reporting. Several studies assessed sample-to-sample parallelism rather than sample-to-standard, contrary to guidelines by regulatory authorities. In conclusion, partial parallelism was infrequently observed and inconsistently reported in most biomarker assays for neurodegeneration. Narrow dilution ranges and variable methodologies limit generalizability. Transparent reporting of dilution protocols and adherence to established analytical validation guidelines are needed. This systematic review has practical implications for clinical trial design, regulatory approval processes, and the reliability of biomarker-based diagnostics.